We have cloned attachment sites of phage lambda on small plasmids. Such DNA molecules were used as the substrates for the intermolecular Int-promoted recombination reaction of phage lambda in vitro. With this system we found that the phage attachment site (att P) and only att P, has to be on a supercoiled DNA molecule for an efficient reaction to take place. We generated shortened att P DNA and recloned such DNA fragments. By characterizing these att DNAs we found that about 200 base pair long sequence is involved in the specific recognition of att P. Further, those att fragments which have lost portions of att P sequence still can act as bacterial att site or pro att sites.